The methylenedioxy group is a chemical motif found in many drugs and natural products. In nature, it is biosynthesized by cytochrome P450 enzymes. This proposal focuses on the use of directed evolution techniques to create a family of cytochrome P450 enzymes that catalyze installation of methylenedioxy bridges in diverse chemical contexts. For this purpose, we propose to use the highly robust and adaptable enzyme CYP102A1, also known as P450BM3. This enzyme has been the focus of many mechanistic, structural, and directed evolution studies. In the latter area, there have been many successful efforts to alter P450BM3's substrate selectivity, and it has proven to be highly adaptable, catalyzing hydroxylation of many different substrates. Mechanistically, methylenedioxy bridge formation is proposed to be mediated via P450- mediated hydroxylation of a methoxy group to yield a hemiformal, followed by attack of the ortho hydroxyl group to afford the bridged product. Consequently, we propose to evolve P450BM3 to catalyze the above reaction sequence. Our specific aims are: (1) To evolve existing P450BM3 variants to catalyze methylenedioxy formation (2) To extend the substrate selectivity of bridge forming enzymes to synthesize novel methylenedioxy-containing intermediates (3) To introduce the evolved enzymes into suitable hosts for the production of methylenedioxy-containing compounds in vivo. To facilitate the above aims, we will take advantage of a large number of existing P450BM3 variants in the Arnold lab. Additionally, we have proposed several high- throughput screens to rapidly identify the desired P450BM3 variants. To synthesize novel methylenedioxy derivatives, we will take advantage of commercially available compounds containing a 1-hydroxy-2-methoxy arrangement. Finally, to create a method for the in vivo synthesis of methylenedioxy-containing compounds, we will construct a pathway from ferulic and vanillic acids to the 2-methoxyphenols, which can then be converted to the desired products by coexpression with the evolved P450BM3 catalysts. PUBLIC HEALTH RELEVANCE: Methylenedioxy is a chemical group found in several existing drugs. Its use in drugs depends on the cost and ease of incorporation. The proposed research is expected to make this chemical group easier to incorporate into drugs, which should lead to more effective drugs available at reduced cost.